8 research outputs found
Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress
The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5′-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3′. Using surface plasmon resonance experiments and electrophoretic mobility shift assays it is demonstrated that Haa1 interacts with high affinity (KD of 2 nM) with the HRE motif present in the ACRE region of TPO3 promoter. No significant interaction was found between Haa1 and HRE motifs having adenine nucleotides at positions 6 and 8 (KD of 396 and 6780 nM, respectively) suggesting that Haa1p does not recognize these motifs in vivo. A lower affinity of Haa1 toward HRE motifs having mutations in the guanine nucleotides at position 7 and 9 (KD of 21 and 119 nM, respectively) was also observed. Altogether, the results obtained indicate that the minimal functional binding site of Haa1 is 5′-(G/C)(A/C)GG(G/C)G-3′. The Haa1-dependent transcriptional regulatory network active in yeast response to acetic acid stress is proposed
Functional characterization of the copper transcription factor AfMac1 from Aspergillus fumigatus
Transcriptional Activation in Yeast in Response to Copper Deficiency Involves Copper-Zinc Superoxide Dismutase*S⃞
Copper is an essential trace element, yet excess copper can lead to
membrane damage, protein oxidation, and DNA cleavage. To balance the need for
copper with the necessity to prevent accumulation to toxic levels, cells have
evolved sophisticated mechanisms to regulate copper acquisition, distribution,
and storage. In Saccharomyces cerevisiae, transcriptional responses
to copper deficiency are mediated by the copper-responsive transcription
factor Mac1. Although Mac1 activates the transcription of genes involved in
high affinity copper uptake during periods of deficiency, little is known
about the mechanisms by which Mac1 senses or responds to reduced copper
availability. Here we show that the copper-dependent enzyme Sod1
(Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1
function in the activation of Mac1 in response to an external copper
deficiency. Genetic ablation of either CCS1 or SOD1 results
in a severe defect in the ability of yeast cells to activate the transcription
of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1
activation and promotes a regulated increase in binding of Mac1 to copper
response elements in the promoter regions of genomic Mac1 target genes.
Although there is precedent for additional roles of Sod1 beyond protection of
the cell from oxygen radicals, the involvement of this protein in
copper-responsive transcriptional regulation has not previously been observed.
Given the presence of both Sod1 and copper-responsive transcription factors in
higher eukaryotes, these studies may yield important insights into how copper
deficiency is sensed and appropriate cellular responses are coordinated